Downstream processing of expressed recombinant proteins is an important consideration in the utilization of cloned gene products. Specifically, purification at industrial levels must supply a protein of sufficient purity in a cost effective manner.
Protein A is a well-known, useful protein originally derived from the cell wall of Staphylococcus aureus. It has the ability to bind the Fc region of most mammalian antibodies of class IgG. Commercial applications of protein A utilizing this property include large-scale affinity purification of monoclonal antibodies, many of which will have therapeutic applications; therapeutic plasma exchange, in which the circulating immune complexes from blood plasma are removed via binding to immobilized protein A; as well as numerous laboratory applications based on immunochemical or histochemical procedures.
To meet these commercial demands, protein A must be economically produced in large quantities and, for therapeutic applications, the protein A must be greater than 99% pure as determined by a variety of assay methods including SDS polyacrylamide gel electrophoresis (PAGE) and size-exclusion HPLC. In addition, contaminating endotoxin levels, especially important in protein A preparations produced from Gram-negative hosts, must be very low, on the order of &lt;1.0 endotoxin units (E.U.)/mg protein A as determined by the gel clot assay (LAL).
There are currently two published methods used to purify protein A. One method uses an IgG affinity column, and the other is a multistep, small scale process which includes the use of a diethylaminoethyl (DEAE) ion-exchange column followed by the use of a sizing column.
The first method utilizes the binding of protein A to the Fc region of the IgG molecule. As developed by Hjelm et al. (Hjelm, H., Hjelm, K., Sjoquist, J [1972] FEBS Letters 28:73-76), IgG is bound to an insoluble matrix which is then packed into a column. A protein A-containing solution is passed over the column at neutral pH in 0.1M H.sub.2 PO.sub.4.sup.-. Under these conditions protein A is selectively removed from the solution due to its interaction with the immobilized IgG. The protein A is eluted from the column with 0.1M glycine-HCl, pH 3.0 and comes off with the buffer front.
Protein A also has been purified using a multistep procedure (Sjoquist, J., Meloun, B., Hjelm, H., [1972] Eur. J. Biochem. 29:572-578). S. aureus, strain Cowan I (NCTC 8530, National Collection of Type Cultures, Central Public Health Laboratory, London, England) is pelleted and resuspended in 0.05M TRIS-HCl [TRIS=Tris(hydroxymethyl)aminomethane], 0.145M NaCl pH 7.5 and heated to 37.degree. C. Lysostaphin and DNAase are added and digestion allowed to go to completion as monitored by absorbance at 520 nm. After the absorbance values become constant, the suspension is centrifuged and the supernatant is treated with 5N HCl to lower its pH to 3.5. The resulting precipitate is removed by centrifugation and the supernatant adjusted to pH 7.0 with 5N NaOH. Ammonium sulfate is added to make an 80% saturated solution. This material is centrifuged and the precipitate removed, redissolved in 0.1M ammonium bicarbonate, pH 8.0, and applied to a DEAE-SEPHADEX (SEPHADEX is a tradename of Pharmacia Inc., Piscataway, N.J.) column equilibrated in 0.1M ammonium bicarbonate, pH 8.0. Under these conditions, the protein A molecule will bind to the positively charged DEAE groups on the column. Protein A is then eluted by increasing the ionic strength of the column buffer via a linear gradient to 0.4M ammonium bicarbonate, pH 8.0.
The protein A removed from the column is concentrated and applied to a SEPHADEX G-100 column equilibrated in 0.05M Tris.HCl, 1.0M NaCl pH 8.0. On such a column proteins are separated according to size--larger molecules are excluded from the interior of the porous SEPHADEX.RTM. and therefore pass through the column more rapidly than smaller molecules which can enter the column packing.
While these purification schemes give satisfactory results for laboratory use, they are inadequate for industrial-scale applications. The problems associated with the first procedure include the high cost of preparation and maintaining an immobilized IgG column. The IgG must be isolated before any work toward purifying protein A can begin. Also, the immobilization of IgG may involve the use of a highly toxic material, cyanogen bromide. Another problem occurs because a biological molecule is part of the column packing. This means that much care must be taken to prevent its contamination or degradation and concomitant loss of binding activity. In spite of such measures, column life is considerably less than that of other properly maintained column packings.
The second purification scheme, discussed above, also is limited for large-scale work due to several reasons. This is a multistep procedure involving acid precipitation, ammonium sulfate precipitation, and buffer exchange before reaching the ion-exchange and sizing column steps. Because each step of a purification results in some loss of material, the overall number of steps should be kept to a minimum. Another drawback to this method is the amount of ammonium bicarbonate used during the ion-exchange chromatography. The cost of using a gradient from 100 mM to 400 mM ammonium bicarbonate would be a substantial portion of the purification cost. The most serious drawback of the procedure, however, is the use of a sizing column. While such columns are suitable for laboratory operations, use of a sizing column at industrial levels is undesirable because capacity is very low (sample size is limited to 2-5% of the column volume), processing time is quite slow compared to other chromatographic methods, and, finally, sizing columns are often troublesome sources of contaminating endotoxins due to bacterial growth in the column.
Finally, both procedures, discussed above, involve the use of pH 3.0-3.5 solutions. This is not suitable for use in the purification of recombinant protein A expressed in E. coli as these organisms contain proteases which will degrade protein A at low pH's.
Thus, there is a need in the art for an efficient process to purify protein A-containing solutions on an industrial scale to obtain high-purity protein A preparations with acceptable low levels of endotoxin. The need is particularly great in the recombinant DNA art of producing protein A, as shown above.